In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity

Abstract

The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer-TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer-TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer-TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer-TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.

Figure 1. Increasing densities of TLR-7/8a arrayed on polymer carriers is associated with particle formation and enhanced lymph node cytokine production

(a) Poly-7/8a were generated by reacting linear biocompatible polymers with nucleophilic TLR-7/8a. (b) Combinatorial synthesis was used to generate Poly-7/8a with varying linker group composition and TLR-7/8a density. The density of agonist arrayed on the polymers (mol% 7/8a) is reported as the percentage of monomers that are linked to TLR-7/8a (e.g., 10 mol% 7/8a indicates 10 out of 100 monomers are linked to TLR-7/8a). (c) A combinatorial library of Poly-7/8a (12.5 nmoles) were subcutaneously administered into hind footpads of mice. After 24 h, lymph nodes draining the site of immunization were harvested and processed to generate a cell suspension that was cultured for 8 h and then evaluated for the presence of IL-12p40 (n = 4). (d) Controls or Poly-7/8a with increasing densities of TLR-7/8a (normalized to 12.5 nmoles TLR-7/8A) were evaluated for particle formation by dynamic light scattering (n = 3) and the capacity to induce IP-10 (n = 6) and IL-12p40 (n = 6) cytokine production in draining lymph nodes at 24 h and 96 h, respectively, after administration. (e) Size distribution plots from dynamic light scattering are shown for selected samples; a confocal microscopy image is shown for a Poly-7/8a with 10 mol% 7/8a that forms particles in aqueous conditions. (f) Data from panel (d) for which Poly-7/8a with 1–2 mol% 7/8a that exist as unimolecular polymer coils (PC-7/8a) or Poly-7/8a with 8–10 mol% 7/8a that exist as submicron polymer particles (PP-7/8a) are grouped together to correlate the effect of Poly-7/8a morphology with lymph node IP-10 (n = 12) and IL-12p40 (n = 12). In vivo screens are representative of two independent experiments. Data on linear axes are reported as mean ± SEM. Data on log scale are reported as geometric mean with 95% Confidence Interval (CI). Comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; Student’s T test was used for comparison of 2 groups; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. SM = small molecule; PC = polymer coil; PP = polymer particle.

Figure 2. Particle formation by Poly-7/8a enhances local retention and promotes uptake by migratory APCs

Dye-labeled particle-forming Poly-7/8a (PP-7/8a), polymer coil Poly-7/8a (PC-7/8a) and small molecule TLR-7/8a (SM 7/8a) were normalized for TLR-7/8a dose (62.5 nmoles) and administered subcutaneously to the left hind footpad of mice (see supplementary Fig. 5a for material composition and doses). (a, b) mice (n = 3) received Infrared (IR) dye-labeled materials and were imaged by X-ray and epifluorescence. (a) Representative images illustrate biodistribution over the first 5 days; (b) amount of TLR-7/8a (nmoles) in draining lymph nodes (n = 3) was estimated from epifluorescence images. (c–f) For mice that received Alexa Fluorophore 488 (AF488) labeled materials, draining lymph nodes were harvested at serial timepoints and evaluated by confocal microscopy (n = 3) or were processed to generate cell suspensions that were evaluated by flow cytometry (n = 3). (c) Representative confocal microscopy images are shown for the groups at 1, 4 and 8 days after administration of adjuvant, with the total fluorescence per imaged section volume quantified and compared across all samples (n = 3). (d) Flow cytometry analysis of the number of total CD11c⁺ DCs and macrophages/monocytes in lymph nodes (n = 3), as well as (e) percent adjuvant uptake (% AF488⁺, n = 3) and (f) the relative amount of adjuvant taken up on a per cell basis (AF488 MFI, n = 3). Whole animal imaging and flow cytometry data are representative of two independent experiments and confocal images from each group are representative of 3 replicates. All data are reported as mean ± SEM; statistical significance is shown for comparisons between PP-7/8a and SM 7/8a and was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Figure 3. Particle forming Poly-7/8a induce high magnitude and persistent local innate immune activation that is associated with enhanced CD8 T cell responses and Th1 skewed antibody responses

Poly-7/8a or controls normalized for TLR-7/8a dose (62.5 nmoles), or Poly-7/8a delivered at different doses (1.25 to 62.5 nmoles), were administered subcutaneously to the left hind footpad of mice. (a–b) Draining lymph nodes were harvested at serial timepoints thereafter and were processed to generate cell suspensions that were evaluated by flow cytometry. (a) CD11c⁺ DCs (n = 3) and (b) macrophages/monocytes (n = 3) were evaluated for their expression of the co-stimulatory molecule CD80. (c, d) Lymph nodes draining the site of administration were harvested at serial timepoints after administration and were processed to generate a cell suspension that was cultured 8 h and then evaluated for the presence of IL-12p40 (n = 4). (e–i) Poly-7/8a, SM 7/8a and controls were formulated with 50 μg of OVA in PBS and given subcutaneously to C57BL/6 mice at days 0 and 14 (see supplementary figure 8a for doses). At day 28, tetramer⁺ CD8 T cell responses were evaluated from whole blood of mice that received either (e) different formulations of TLR-7/8a (n = 5) or (f) different doses or potencies of particle-forming Poly-7/8a (PP-7/8a) admixed with OVA (n = 5). Serum from vaccinated mice was evaluated for (g) anti-OVA total IgG antibody titers (n = 5), as well as (h, i) the ratio of Ig1/IgG2c isotypes (n = 5). All data are representative of two or more independent experiments. Data on log scale are reported as geometric mean with 95% CI. Comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Figure 4. Persistent, locally restricted innate immune activation is necessary and sufficient for eliciting protective CD8 and Th1-type CD4 T cell responses

(a–c) CpG ODN 1826 (3.1 nmoles, 20 μg), R848 (62.5 nmoles, 20 μg) or PP-7/8a (62.5 nmoles, 120 μg) were delivered subcutaneously into both hind footpads of C57BL/6 mice. (a) Supernatant of ex vivo cultured lymph node cell suspensions (n = 4) and (b) serum (n = 5) were assessed for IL-12p40 by ELISA at serial timepoints. (c) Percent body weight change (n = 3) following subcutaneous administration of different vaccine adjuvants (significance is shown for comparison with naïve; two-way ANOVA with Bonferroni correction). (d) Relationship between biodistribution and local and systemic innate immune activation. (e, f) C57BL/6 mice received subcutaneous administration of protein antigen (either 50 μg of OVA, or 20 μg of SIV Gag p41) formulated with adjuvant at days 0 and 14. At day 24, tetramer⁺ CD8 T cell responses were assessed from whole blood by flow cytometry (n = 6). (g, h) Mice were challenged intravenously at day 28 with either (g) LM-OVA or (h) LM-Gag, and bacterial burden in spleens (n = 6) was evaluated on day 31 and 30, respectively. (i–l) C57BL/6 mice received subcutaneous immunizations of 20 μg of MML with or without adjuvant on days 0, 21 and 42. (i) Splenocytes were isolated on day 70 and stimulated in vitro with an MML peptide pool. CD4 T cells in the mixed splenocyte cultures were evaluated for Th1 characteristic cytokine (IFNγ, IL-2 and TNFα) production (n = 4). (j) Mice were challenged intradermally in both ears with L. major at day 70. Ear lesion diameters (n = 6) were measured for 12 weeks (significance is shown for comparison with protein alone). All data are representative of two or more independent experiments, except the Leishmania ear lesion kinetic is from a single study. Data on log scale are reported as geometric mean with 95% CI. Unless stated otherwise, comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Figure 5. Thermo-responsive polymer particles (TRPP) permit temperature-dependent particle assembly that leads to persistent innate immune activation and protective CD8 T cell responses

(a) Schematic of TRPP shown reversibly assembling into particles. (b) Transition temperatures (TT) were empirically determined by measuring the turbidity (OD at 490 nm) of solutions of TRPP in PBS over a range of temperatures. (c) Table summarizing the thermo-responsive properties of select TRPP. (d) TRPP-7/8a and a TRPP control were delivered subcutaneously into both hind footpads of C57BL/6 mice. Popliteal lymph nodes draining the vaccination site were harvested at 24 h (n = 6) and 72 h (n = 4) and then processed to create a cell suspension that was cultured ex vivo for 8h and then evaluated for the presence of IL-12p40 and IP-10 by ELISA. (e, f) C57BL/6 mice (n = 5) received subcutaneous administration of 50 μg of OVA alone or admixed with adjuvant at days 0 and 14. (e) Tetramer⁺ CD8 T cell responses were evaluated at day 24 (n = 5). (f) Mice were challenged intravenously at day 28 with LM-OVA and bacterial burden in spleens was evaluated on day 31 (n = 5). All data are representative of two independent experiments. Data on log scale are reported as geometric mean with 95% CI. Comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01.

Figure 6. Co-delivery of TLR-7/8a and protein antigen on a self-assembling thermo-responsive vaccine particle

(a) Cartoon schematic of a thermo-responsive Poly-7/8a (TRPP-7/8a) modified with a coil peptide that forms heterodimers with a recombinant HIV Gag-coil fusion protein to form TRPP-7/8a-(CC)-Gag. Heterodimerization occurs at room temperature and particle formation results at temperatures greater than 33°C. (b) Temperature-dependent particle formation illustrated by dynamic light scattering. (c) Aqueous solutions of TRPP-7/8a-(CC)-Gag at 25°C and 37°C. (d, e) Co-localization of HIV Gag (labeled with anti-Gag PE) with TRPP-7/8a (labeled with carboxyrhodamine 110) was confirmed by (d) flow cytometry and (e) confocal microscopy. (f–i) BALB/c mice received subcutaneous administration of 50 μg of HIV-Gag coil formulated with either a control or TRPP-7/8a normalized for TLR-7/8a dose (1x dose = 2.5 nmoles, or 3x dose = 7.5 nmoles) at days 0 and 14. At day 28, DLN, spleen and serum from vaccinated mice were collected for analysis. Splenocytes were stimulated in vitro with an HIV Gag peptide pool. Antigen-specific IFNγ-producing (f) CD4 T cells (n = 5) and (g) CD8 T cells (n = 5) in the mixed splenocyte cultures, as well as (h) Tfh cells (n = 5) in draining lymph nodes were quantified by flow cytometry. (i) Serum was evaluated for anti-HIV Gag total IgG antibody titers (n = 5). In vivo studies are representative of two independent experiments. Data on linear axes are reported as mean ± SEM. Data on log scale are reported as geometric mean with 95% CI. Comparison of multiple groups for statistical significance was determined using Kruskal-Wallis ANOVA with Dunn’s post test; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. CC = coiled-coil interaction.